Gene transcription is the tightly controlled first step in gene expression. In fungi, only one conserved pathway exists for synthesizing mRNA. mRNA synthesis in fungi is carried out in the nucleus only by RNA polymerase II (Pol II), in conjunction with over 20 additional polypeptides that include DNA binding proteins, protein kinase, multiple DNA unwinding activities, and processivity and assembly factors (FIG. 1). Factors involved in fungal transcription differ significantly from the factors controlling human mRNA transcription. For example, the process of transcription initiation differs between yeast and human cells (FIG. 2). A number of exploitable differences between fungal and human nuclear polymerase involved in transcription have been determined. Because gene transcription is highly conserved among fungi and is distinct from mammalian transcription, the present inventors expected inhibitors of fungal RNA polymerase II to be broad spectrum and non-cytotoxic. Due to the uniqueness of the molecular target, fungal transcription inhibitors were also expected to be effective against fungi with acquired resistance to marketed antifungal with different mechanisms of action.
Accordingly, the present inventors have sought out compounds which are able to inhibit fungal transcription processes by inhibition of RNA polymerase II. Pol II consists of 11-12 subunits of which 4 are shared with fungal DNA-dependent RNA polymerase I and III. Pol II was chosen as an antifungal drug discovery target because of its essential role in fungal gene transcription. RNA polymerase II was purified from Candida albicans, Aspergillus nidulans and Saccharomyces cerevisiae and from human cells. A robust, reproducible high throughput RNA elongation assay using C. albicans RNA polymerase II was developed and several novel antifungal agents were identified as potent inhibitors of fungal RNA polymerase II, selective for fungal polymerase IIs relative to human RNA polymerase II.
Screening and counterscreening of a small molecule chemical library have produced a number of potent Pol II inhibitors. Certain of these inhibitors are rapidly fungicidal, with broad spectrum antifungal activity in vitro. Moreover, new compounds which include azo and stilbene analogues of these inhibitors have been prepared which have potent antifungal activity, and which are active against amphotericin- and azole-resistant fungal strains and effective as oral agents in a murine model of systemic C. albicans infection.